The present invention relates to the identification of an isolated, full-length rat nucleic acid molecule encoding an osteoactivin protein, therapeutic compositions comprising an osteoactivin protein, and methods for using the nucleic acid molecules and proteins for stimulating bone differentiation. The invention also relates to methods for treating bone disorders, including osteopetrosis and osteoporosis.
The formation and maintenance of the vertebrate skeleton requires the interactions of many cell types and growth factors and other molecules. The past decade has witnessed an explosive growth in the general understanding of growth factors and other proteins that mediate the complex coordination of bone formation and bone resorption by these different cell types in skeletal modeling and remodeling (Popoff and Marks, Oral and Maxillofacial Clinics of North America 9:563-579 (1997)).
In general, the bone remodeling cycle involves a complex series of sequential steps that are highly regulated. The initial xe2x80x9cactivationxe2x80x9d phase of bone remodeling begins early in fetal life and is dependent on the effects of local and systemic growth factors on mesenchymal cells of the osteoblast lineage (Eriksen, Endocrinol. Rev. 7:379-408 (1986)). These cells interact with hematopoietic precursors to form osteoclasts in the xe2x80x9cresorptionxe2x80x9d phase. This leads to the differentiation, migration and fusion of the large multinucleated osteoclasts. These cells attach to the mineralized bone surface and initiate resorption by the secretion of hydrogen ions and lysosomal enzymes. Osteoclastic resorption produces irregular scalloped cavities on bone surface. Once the osteoclasts have completed their work of bone removal, there is a xe2x80x9creversalxe2x80x9d phase during which mononuclear cells, which may be of the macrophage lineage, are present on the bone surface. These cells further degrade collagen, deposit proteoglycans, and release growth factors that signal the initiation of the xe2x80x9cformationxe2x80x9d phase. During the final formation phase of the remodeling cycle, the cavity created by resorption can be completely filled in with successive layers of osteoblasts, which differentiate from their mesenchymal precursors and lay down a mineralizable matrix. (Raisz, Clin. Chem. 45:1353-1358 (1999)).
With bone disorders associated with decreased bone mass, osteoclastic resorption outweighs osteoblastic bone formation, resulting in bone loss. While treatments that stimulate bone formation would be beneficial in treating or preventing bone loss, current therapies are suboptimal (Canalis, J. Clin. Invest. 106:177-179 (2000); Raisz, J. Bone Min. Metab 17:79-89 (1999)).
An animal model useful in bone studies is the osteopetrosis (op) mutation in the rat. Osteopetrosis describes a group of congenital bone disorders that are characterized by a generalized increase in skeletal mass resulting from a primary defect in osteoclast-mediated bone resorption (Popoff and Schneider, Molec. Med. Today 2:349-358 (1996)). Numerous osteopetrotic mutations have been described in other species, including human and mouse. The bone that is formed as the skeleton develops and grows in animals with this mutation is not resorbed, resulting in the failure to develop bone marrow cavities. The osteopetrotic mutations are pathogenetically heterogeneous since the point at which osteoclast development or activation is intercepted differs for each mutation (Popoff and Marks, Bone 17:437-445 (1995)). Although osteoclast hypofunction is universal among the osteopetrotic mutations, genetic abnormalities involving osteoblast development/function (i.e., bone formation), mineral homeostasis and the immune and endocrine systems have also been reported within this disorder (Seifert et al., Clin. Orthop. 294:23-33 (1993)).
To date, pharmaceutical approaches to managing osteoporosis or osteopetrosis are of limited effectiveness. Therefore, alternative therapies are needed to modulate bone cell differentiation and bone formation, and to treat bone disorders such as osteoporosis and osteopetrosis.
The present invention is based, in part, on the discovery of a novel rat gene encoding an osteoactivin protein. The nucleotide sequence of full-length cDNA of the gene is shown in SEQ ID NO:1. The nucleotide sequence of the cDNA encoding the osteoactivin protein is shown in nucleotides 115 to 1,830 of SEQ ID NO:1 and the corresponding amino acid sequence of the osteoactivin protein is shown in SEQ ID NO:2. The polynucleotide sequence of the cDNA encoding the osteoactivin protein lacking the signal sequence is shown in nucleotides 181-1830 of SEQ ID NO:1 and the corresponding osteoactivin polypeptide lacking the signal sequence is from amino acid residues 23-572 of SEQ ID NO:2. The claimed invention also relates to antibodies which recognize one or more epitopes of the osteoactivin protein. The claimed invention provides therapeutic compositions comprising (i) a nucleic acid molecule encoding an osteoactivin protein, (ii) an osteoactivin protein, or (iii) an antibody to an osteoactivin protein. These therapeutic compositions are useful to treat bone disorders and to stimulate bone formation and bone cell differentiation.
Accordingly, in one aspect, the invention is directed to molecules encoding an osteoactivin protein. One embodiment of this aspect is a nucleic acid molecule encoding a rat osteoactivin protein having a molecular weight of 63.8 kilodaltons (kD), wherein said osteoactivin protein stimulates bone cell differentiation. In a related embodiment of this aspect, the invention encompasses a full-length nucleic acid molecule which encodes an osteoactivin protein, wherein said nucleic acid comprises the nucleic acid sequence of SEQ ID NO:1. In another embodiment, the invention provides a nucleic acid molecule encoding an osteoactivin protein, wherein said nucleic acid hybridizes to the complement of SEQ ID NO:1 under moderately stringent conditions. In a preferred embodiment, the nucleic acid molecule encoding an osteoactivin protein having at least 92% sequence identity with the nucleic acid sequence of SEQ ID NO:1 is described. In some embodiments, the nucleic acid molecule encodes a polypeptide comprising SEQ ID NO:2. The invention also embodies the nucleic acid molecule encoding an osteoactivin polypeptide comprising amino acid residues 23-572 of SEQ ID NO:2. In other embodiments, the invention provides a nucleic acid encoding an osteoactivin protein, wherein said nucleic acid comprises from nucleotide 115 to nucleotide 1,830 of SEQ ID NO:1. Other embodiments of the invention provide a polynucleotide encoding an osteoactivin protein lacking the leader sequence, wherein said polynucleotide comprises from nucleic acid residues from 181 to1830 of SEQ ID NO:1. In still other embodiments, the invention provides a nucleic acid encoding an osteoactivin protein, wherein said nucleic acid molecule hybridizes to the complement of nucleotide 115 to nucleotide 1,830 of SEQ ID NO:1 under moderately stringent conditions. In yet another embodiment of this aspect, the invention further provides a nucleic acid molecule encoding an osteoactivin protein having at least 92% sequence identity with the nucleic acid sequence from nucleotide 115 to nucleotide 1,830 of SEQ ID NO:1.
As used herein, the term xe2x80x9cnucleic acid moleculexe2x80x9d includes DNA molecules (e.g., a cDNA or genomic DNA) and RNA molecules (e.g., an mRNA) and analogs of the DNA or RNA generated, e.g., by the use of nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA. Nucleic acid molecules include naturally occurring nucleic acid molecules which are separated from other molecules which are present in the natural source of the nucleic acid. For example, a nucleic acid molecule includes genomic DNA which is separated from the chromosome with which the genomic DNA is naturally associated. Preferably, a naturally occurring nucleic acid molecule is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5xe2x80x2 and/or 3xe2x80x2 ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than 5 kilobases (kb), 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of 5xe2x80x2 and/or 3xe2x80x2 nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an isolated nucleic acid molecule, such as a cDNA molecule, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
As used herein, the term xe2x80x9costeoactivin proteinxe2x80x9d refers to a protein including the amino acid sequence of SEQ ID NO:2, the murine osteoactivin protein homolog, nmb, of SEQ ID NO:5, the human osteoactivin protein homolog, nmb, of SEQ ID NO:6, and the amino acid sequence comprising amino acid residues 23-572 of SEQ ID NO:2. Further, an osteoactivin protein has at least 50% sequence identity, preferably 70% sequence identity, and more preferably 90% sequence identity to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:5, or SEQ ID NO:6, and stimulates bone cell differentiation or bone formation. Preferably, the osteoactivin protein is naturally occurring in a mammalian species.
As used herein, xe2x80x9cstimulates bone cell differentiationxe2x80x9d means any increase in bone cell number or size, including without limitation, the increase in the rate of bone cell division or precursor bone cell recruitment from the stem cells or bone marrow cells, and an increase in bone cell size. Such bone cell differentiation can be measured by well known cell proliferation assays (e.g., 3H-thymidine incorporation) and bone differentiation assays (e.g., Owen, et al., J. Cell Physiol. 143:420-30 (1990)).
As used herein, by xe2x80x9cstimulates bone formationxe2x80x9d is meant the recruitment of osteoblasts or osteoblast precursors to a bone site, which results in differentiation of the cells into mature osteoblasts and their secretion of collagenous matrix which mineralizes into bone matter and increases bone mass at the site. This term also encompasses the increased production and secretion of collagenous matrix by mature osteoblasts.
As used herein, the term xe2x80x9chybridizes under moderately stringent conditionsxe2x80x9d describes conditions for hybridization and washing. Stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley and Sons, N.Y. (1989), 6.3.1-6.3.6. An example of moderately stringent hybridization conditions is hybridization in 50% formamide 6xc3x97SSC at 42xc2x0 C., followed by one or more washes in 0.2xc3x97SSC, 0.1% sodium dodecyl sulfate (SDS) at 55xc2x0 C. In some preferable embodiments, an isolated nucleic acid molecule of the invention that hybridizes under moderately stringent conditions to the sequence of SEQ ID NO:1 corresponds to a naturally-occurring nucleic acid molecule.
By two nucleic acid molecules being xe2x80x9ccomplementaryxe2x80x9d to one another or hybridizing to a xe2x80x9ccomplementxe2x80x9d of another nucleic acid molecule is meant that the first nucleic acid molecule (e.g., an oligonucleotide) is able to form Watson-Crick base pair hydrogen bonds (i.e., hybridize) with the second nucleic acid molecule to form a duplex.
As used herein, a percent xe2x80x9csequence identityxe2x80x9d refers to a calculation of xe2x80x9chomologyxe2x80x9d or xe2x80x9cidentityxe2x80x9d between two different nucleic acid or amino acid sequences (the terms are used interchangeably herein) when the sequences are aligned and compared. The percent sequence identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
In another aspect of the invention, the invention features an isolated and substantially pure osteoactivin protein, or polypeptide fragment thereof. One preferred embodiment of this aspect of the invention is an isolated and substantially pure rat osteoactivin protein, or polypeptide fragment thereof, wherein said protein comprises the amino acid sequence of SEQ ID NO:2. In another embodiment, the invention provides an isolated and substantially pure, non-human, non-murine osteoactivin protein, or polypeptide fragment thereof, having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:2, wherein said osteoactivin protein or polypeptide fragment thereof stimulates bone cell differentiation or bone formation.
An xe2x80x9cisolatedxe2x80x9d or xe2x80x9cpurifiedxe2x80x9d osteoactivin protein or polypeptide is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. In one embodiment, the language xe2x80x9csubstantially freexe2x80x9d means preparation of osteoactivin protein having less than 30%, 20%, 10% and more preferably less than 5% (by weight), of non-osteoactivin protein (also referred to herein as a xe2x80x9ccontaminating proteinxe2x80x9d), or of chemical precursors or non-osteoactivin compounds. When the osteoactivin protein, or biologically active portion thereof, is recombinantly produced, it is also preferably substantially free of culture medium, i.e., the culture medium represents less than 20%, more preferably less than 10%, and most preferably less than 5% of the volume of the protein preparation. The invention includes isolated or purified preparations of at least 0.01 milligrams, at least 0.1 milligrams, at least 1.0 milligrams, and at least 10 milligrams by weight.
Also included, in another aspect of the invention, are expression vectors containing nucleic acid molecules encoding an osteoactivin protein or polypeptide fragment therein. In one embodiment, the invention features a biologically functional expression vector comprising a nucleic acid sequence encoding an osteoactivin protein, or biologically active polypeptide fragment thereof, wherein said osteoactivin protein comprises the amino acid sequence of SEQ ID NO:2, or has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:2, and which stimulates bone cell differentiation or bone formation.
In another embodiment of this aspect, the invention is directed to biologically functional expression vectors comprising a nucleic acid molecule encoding a rat osteoactivin protein having a molecular weight of 63.8 kD, wherein said osteoactivin protein stimulates bone cell differentiation. In another embodiment, a biologically functional expression vector is provided which comprises the nucleic acid sequence of SEQ ID NO:1. The invention also encompasses a biologically functional expression vector comprising said nucleic acid molecule encoding an osteoactivin protein, wherein the nucleic acid molecule hybridizes to the complement of SEQ ID NO:1 under moderately stringent conditions. The invention also provides a biologically functional expression vector comprising a nucleic acid molecule encoding an osteoactivin protein and having at least 92% sequence identity with the nucleic acid sequence of SEQ ID NO:1. In another embodiment, the invention provides a biologically functional expression vector comprising a nucleic acid molecule encoding an osteoactivin protein, wherein said nucleic acid molecule comprises from nucleotide 115 to nucleotide 1,830 of SEQ ID NO:1. Yet another embodiment of this aspect of the invention includes a biologically functional expression vector comprising a nucleic acid molecule encoding an osteoactivin protein, wherein said nucleic acid molecule hybridizes to the complement of nucleotide 115 to nucleotide 1,830 of SEQ ID NO:1 under moderately stringent conditions. Still yet another embodiment is directed to a biologically functional expression vector comprising said nucleic acid molecule encoding an osteoactivin protein having at least 92% sequence identity with the nucleic acid sequence from nucleotide 115 to nucleotide 1,830 of SEQ ID NO:1. In each of these embodiments, the vector may be a plasmid or a viral vector.
As used herein, the term xe2x80x9cvectorxe2x80x9d refers to a composition capable of carrying a nucleic acid molecule to its target. Vectors include liposomes and nucleic acid molecules capable of transporting another nucleic acid to which it has been linked. Such nucleic acid vectors include plasmids, cosmids, or viral vectors. The nucleic acid vector can be capable of autonomous replication or it can integrate into a host DNA. Viral vectors include, e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses. A xe2x80x9cbiologically functional expression vectorxe2x80x9d as used herein refers to a vector used to incorporate nucleic acid molecules of the invention, including an osteoactivin-encoding nucleic acid, in a form suitable for expression in a host cell.
In yet another aspect, the invention features immunoglobulins such as antibodies and antigen-binding fragments thereof, that recognize and bind one or more epitopes of the osteoactivin proteins or polypeptide fragments thereof. In one embodiment of this aspect, the invention provides a substantially pure antibody that specifically binds to one or more epitopes of an osteoactivin protein, or a polypeptide fragment thereof, wherein said osteoactivin protein stimulates bone cell differentiation. The invention further provides a substantially pure antibody that specifically binds to one or more epitopes of an osteoactivin protein, or a polypeptide fragment thereof, wherein said osteoactivin protein comprises the amino acid sequence of SEQ ID NO:6. In a related embodiment, a substantially pure antibody that specifically binds to one or more epitopes of an osteoactivin protein, or polypeptide fragment thereof, wherein said osteoactivin protein comprises the amino acid sequence of SEQ ID NO:2 is provided. In a preferred embodiment, the antibody is selected from the group consisting of an antibody which binds to one or more epitopes of an osteoactivin peptide 35 having SEQ ID NO:3 and an antibody which binds to one or more epitopes of an osteoactivin peptide 551 having SEQ ID NO:4. In a particularly preferred embodiment, the antibody binds to one or more epitopes of amino acids 538-553 of SEQ ID NO:6. Another preferred embodiment is an antibody which specifically binds to an osteoactivin protein, or polypeptide fragment thereof, having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:2, wherein said osteoactivin protein or polypeptide fragment thereof stimulates bone cell differentiation or bone formation. In each of these embodiments, the antibody may be a polyclonal or a monoclonal antibody.
The term xe2x80x9cantibodyxe2x80x9d as used herein refers to an immunoglobulin molecule or immunologically active portion thereof, i.e., an antigen-binding portion. Examples of immunologically active portions of immunoglobulin molecules include F(ab), Fv, and F(abxe2x80x2)2 fragments which can be generated by cleaving the antibody with an enzyme such as pepsin.
The term xe2x80x9cepitopexe2x80x9d as used herein means that region of amino acid residues of an osteoactivin protein antigen that is specifically recognized by an anti-osteoactivin antibody.
By xe2x80x9cspecifically bindsxe2x80x9d means an antibody that physically interacts with its specific ligand (i.e., an osteoactivin protein or biologically active polypeptide fragment thereof) with greater affinity that it binds to other molecules.
The invention further provides methods for producing a substantially pure osteoactivin protein, or polypeptide fragment thereof, comprising: (a) culturing a cell stably transformed with a gene comprising a nucleic acid molecule encoding an osteoactivin protein, wherein said nucleic acid comprises the nucleic acid sequence from nucleotide 115 to nucleotide 1,830 of SEQ ID NO:1; and (b) isolating and purifying the osteoactivin protein from the culture medium. Another preferred embodiment includes a method for producing a substantially pure osteoactivin protein, or polypeptide fragment thereof, comprising: (a) culturing a cell stably transformed with a gene comprising said nucleic acid molecule encoding an osteoactivin protein having at least 92% sequence identity with the nucleic acid sequence from nucleotide 115 to nucleotide 1,830 of SEQ ID NO:1; and (b) isolating and purifying said osteoactivin protein from said culture medium. A method for producing a substantially pure osteoactivin protein, or polypeptide fragment thereof, comprising: (a) culturing a cell stably transfected with a vector comprising the nucleic acid molecule encoding an osteoactivin protein, wherein said nucleic acid comprises the nucleic acid sequence from nucleotide 115 to nucleotide 1,830 of SEQ ID NO:1; and (b) isolating and purifying said osteoactivin protein from said culture medium, is also provided. In a related embodiment, the invention provides a method for producing a substantially pure osteoactivin protein, or polypeptide fragment thereof, comprising: (a) culturing a cell stably transfected with a vector comprising said nucleic acid molecule encoding an osteoactivin protein having at least 92% sequence identity with the nucleic acid sequence from nucleotide 115 to nucleotide 1,830 of SEQ ID NO:1; and (b) isolating and purifying said osteoactivin protein from said culture medium.
As used herein, the terms osteoactivin xe2x80x9cgenexe2x80x9d and xe2x80x9crecombinant genexe2x80x9d refer to nucleic acid molecules which include an open reading frame encoding an osteoactivin protein, such as a mammalian osteoactivin protein, and can further include non-coding regulatory sequences, and introns. These genes can be isolated from genomic DNA, cloned by recombinant means, or chemically synthesized.
As used herein, the terms xe2x80x9ctransformationxe2x80x9d and xe2x80x9ctransfectionxe2x80x9d are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a prokaryotic or eukaryotic host cell, including, but not limited to, calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation, such that the DNA within the vector is expressed in the host cell.
The invention also provides for therapeutic compositions of the disclosed nucleic acid molecules and osteoactivin proteins and antibodies. Accordingly, in another aspect, the invention provides a therapeutic composition comprising a pharmaceutically acceptable carrier or delivery vehicle and a nucleic acid encoding an osteoactivin protein, or biologically active polypeptide fragment thereof, wherein said osteoactivin protein stimulates bone cell differentiation. In some embodiments, the therapeutic composition comprises a nucleic acid molecule encoding a human osteoactivin or encoding the amino acid sequence of SEQ ID NO:6. In still another embodiment, the invention encompasses a therapeutic composition comprising a nucleic acid molecule encoding an osteoactivin protein having at least 92% sequence identity with the nucleic acid sequence of SEQ ID NO:1, and a pharmaceutically acceptable delivery vehicle.
In certain embodiments of this aspect of the invention, the therapeutic composition further comprises a mediator such as a cytokine or a growth factor. Non-limiting examples of such mediators include interleukin-1, tumor necrosis factor, lymphotoxin, interleukin-6, prostaglandins of the E-series, leukotrienes, lipopolysaccharides, transforming growth factor-xcex2, and colony-stimulating factors. In another related aspect, the invention provides a therapeutic composition comprising an agent that stimulates osteoactivin-mediated bone formation. The term xe2x80x9cmediatorxe2x80x9d refers to a molecule that directly modulates, mediates, or changes the expression of an osteoactivin protein.
As used herein, a xe2x80x9ctherapeutic compositionxe2x80x9d refers to a composition comprising an active ingredient required to cause a desired effect when a therapeutically effective amount of the composition is administered to a mammal in need thereof.
Within the present invention, a xe2x80x9ctherapeutically effective amountxe2x80x9d of a composition is that amount of each active component of the therapeutic composition that is sufficient to show a benefit (e.g., a reduction in a symptom associated with the disorder, disease, or condition being treated). When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the benefit, whether administered in combination, serially, or simultaneously.
As used herein, the term xe2x80x9cpharmaceutically acceptable delivery vehiclexe2x80x9d refers to carriers that do not negatively affect the biological activity of the therapeutic molecule or compound to be placed therein. Preferably, the vehicle targets bone cells. The characteristics of the delivery vehicle will depend on the route of administration. Therapeutic compositions may contain, in addition to the active compound, diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.
Other therapeutic compositions within the scope of this invention cover compositions comprising vectors. Accordingly, in one embodiment, the invention features a therapeutic composition comprising a biologically functional expression vector comprising a nucleic acid sequence encoding an osteoactivin protein, wherein said osteoactivin protein stimulates bone cell differentiation or bone formation, and a pharmaceutically acceptable delivery vehicle. In another embodiment, the invention provides a therapeutic composition comprising a biologically functional expression vector comprising a nucleic acid molecule encoding an osteoactivin protein having at least 92% sequence identity with the nucleic acid sequence of SEQ ID NO:1, and a pharmaceutically acceptable delivery vehicle. In certain embodiments of this aspect of the invention, the therapeutic composition further comprises a mediator, such as interleukin-1, tumor necrosis factor, lymphotoxin, interleukin-6, prostaglandins of the E-series, leukotrienes, lipopolysaccharides, transforming growth factor-xcex2, or colony-stimulating factors, or a nucleic acid molecule encoding any of these mediator polypeptides.
Still other embodiments within this aspect of the invention are directed to therapeutic compositions comprising an osteoactivin protein. Accordingly, the invention features a therapeutic composition comprising a pharmaceutically acceptable carrier or delivery vehicle and an osteoactivin protein, or biologically active polypeptide fragment thereof, wherein said osteoactivin protein stimulates bone cell differentiation or bone formation. In some embodiments, the osteoactivin protein in the therapeutic composition is human. In other embodiments, the osteoactivin protein comprises SEQ ID NO:6. In another embodiment of this aspect, the invention covers a therapeutic composition comprising an osteoactivin protein, or polypeptide fragment thereof, wherein said protein comprises the amino acid sequence of SEQ ID NO:2, and a pharmaceutically acceptable delivery vehicle. In yet a further embodiment, a therapeutic composition comprising a pharmaceutically acceptable delivery vehicle and a non-human, non-murine osteoactivin protein, or polypeptide fragment thereof, having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:2, wherein said osteoactivin protein or polypeptide fragment thereof stimulates bone cell differentiation or bone formation. In certain embodiments of this aspect of the invention, the therapeutic composition may further comprises a mediator, including interleukin-1, tumor necrosis factor, lymphotoxin, interleukin-6, prostaglandins of the E-series, leukotrienes, lipopolysaccharides, transforming growth factor-xcex2, and colony-stimulating factors. In another aspect, the invention provides a therapeutic composition comprising an agent that inhibits osteoactivin-mediated bone formation, and a pharmaceutically acceptable delivery vehicle.
As used herein, a xe2x80x9cbiologically active portionxe2x80x9d of an osteoactivin protein includes a fragment of an osteoactivin protein which is capable of affecting bone differentiation or bone formation.
Additional therapeutic compositions of the invention relate to those comprising antibodies that react with, or specifically bind, osteoactivin proteins. In one embodiment of this aspect, the invention provides a therapeutic composition comprising a pharmaceutically acceptable delivery vehicle and an antibody that specifically binds to one or more epitopes of an osteoactivin protein, or polypeptide fragment thereof, wherein said osteoactivin protein stimulates bone differentiation or bone formation. In a related embodiment, the invention covers a therapeutic composition comprising a substantially pure antibody that specifically binds to one or more epitopes of an osteoactivin protein, or polypeptide fragment thereof, wherein said osteoactivin protein comprises the amino acid sequence of SEQ ID NO:2, and a pharmaceutically acceptable delivery vehicle. In a preferred embodiment, the antibody of a therapeutic composition is selected from the group consisting of an antibody which binds to one or more epitopes of an osteoactivin peptide 35 having SEQ ID NO:3, an antibody which binds to one or more epitopes of an osteoactivin peptide 551 having SEQ ID NO:4, and an antibody which binds to one or more epitopes of amino acids 538-553 of the human osteoactivin protein of SEQ ID NO:6, together with a pharmaceutically acceptable delivery vehicle. Another preferred antibody, according to this aspect of the invention, is one in which the antibody of the therapeutic composition specifically binds to a non-human, non-murine osteoactivin protein, or polypeptide fragment thereof, having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:2, wherein said osteoactivin protein or polypeptide fragment thereof stimulates bone cell differentiation or bone formation, together with a pharmaceutically acceptable delivery vehicle. In certain embodiments, the antibody of the invention is a polyclonal or a monoclonal antibody.
In yet another aspect, the invention provides in vivo methods of stimulating bone formation in a mammal, comprising administering to said mammal a therapeutically effective amount of a nucleic acid molecule encoding an osteoactivin protein or peptide thereof, or an osteoactivin protein, or biologically active polypeptide fragment thereof, or an agent that enhances osteoactivin-mediated bone cell differentiation or bone formation. In other embodiments of this aspect, ex vivo methods for stimulating bone formation in a human are described, comprising the steps of: (a) extracting osteoblast cells from said human; (b) contacting said osteoblast cells with a therapeutically effective amount of a nucleic acid molecule encoding an osteoactivin protein, or an osteoactivin protein, or biologically active polypeptide fragment thereof; and (c) returning said cells to the bone of said human.
xe2x80x9cMammalxe2x80x9d as used herein means any animal classified as a mammal including humans, cows, horses, dogs, mice, cats, goats, pigs, and sheep.
In another aspect, the invention features in vivo methods for inhibiting bone formation in a mammal, comprising administering to said mammal a therapeutically effective amount of any of the therapeutic compositions of the present invention comprising antibodies. In a related aspect, the invention also provides a method for inhibiting bone formation in a mammal, comprising administering to said mammal a therapeutically effective amount of an agent that inhibits osteoactivin-mediated bone formation.
In another aspect, the invention provides in vivo methods of inhibiting bone formation or bone cell differentiation in a mammal, comprising administering to said mammal a therapeutically effective amount of any of the therapeutic compositions of the invention comprising, in part, an antibody.
The invention provides, in yet another aspect, in vivo methods of treating bone disorders in a mammal, such as a human, comprising administering to said mammal a therapeutically effective amount of any of the therapeutic compositions of the invention, comprising an anti-osteoactivin antibody or an agent that inhibits osteoactivin-mediated bone differentiation. In a related embodiment, the invention provides ex vivo methods for treating bone disorders in a mammal, comprising the steps of: (a) extracting osteoblast cells from said mammal; (b) contacting said osteoblast cells with a therapeutically effective amount of an antibody specific for osteoactivin protein or an agent that inhibits osteoactivin-mediated bone cell differentiation or bone formation; and (c) returning said contacted cells to the bone of said mammal. In preferred embodiments, the bone disorder treated by the method is selected from the group consisting of an ectopic bone formation, osteoporosis, periodontal disease, and osteopetrosis.
The phrase xe2x80x9cbone disorder,xe2x80x9d as used herein, refers to a pathological disorder, disease, or condition in a mammal in which there is an imbalance in the ratio of bone formation to bone resorption, such that, if left untreated, would result in that mammal exhibiting an abnormal mass of bone.
xe2x80x9cTreating,xe2x80x9d xe2x80x9ctreatment,xe2x80x9d and xe2x80x9ctherapy,xe2x80x9d as used herein, refer to curative, prophylactic, or preventative manipulations, or manipulations which stimulate bone cell differentiation or bone formation, postpone the development of bone disorder symptoms, and/or reduce the severity of bone disorders and/or such symptoms that will or are expected to develop from a bone disorder. The terms further include ameliorating existing bone disorder symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, preventing or reversing metabolic causes of symptoms, preventing or reversing bone growth, and/or encouraging bone resorption. Thus, the terms denote that a beneficial result has been conferred on a mammal with a bone disorder, or with the potential to develop such disorder.
In another aspect, the invention provides in vivo methods of treating bone disorders in a mammal such as a human, comprising administering to said mammal, a therapeutically effective amount of any of therapeutic compositions of the invention comprising a nucleic acid molecule encoding an osteoactivin protein, or an osteoactivin protein, or biologically active polypeptide fragment thereof, wherein said osteoactivin protein or biologically active polypeptide fragment thereof stimulates bone formation or bone cell differentiation. In other embodiments of this aspect, ex vivo methods for treating bone disorders in a mammal are provided. These methods comprise the steps of: (a) extracting osteoblast cells from said mammal; (b) contacting said osteoblast cells with a therapeutically effective amount of any of the therapeutic compositions comprising a nucleic acid molecule encoding an osteoactivin protein, or biologically active polypeptide fragment thereof, of the invention; and (c) returning said cells to the bone of said mammal, wherein said osteoactivin protein or biologically active polypeptide fragment thereof stimulates bone formation or bone cell differentiation. In certain embodiments, the bone disorder treated is selected from the group consisting of an ectopic bone formation, osteoporosis, periodontal disease, and osteopetrosis. In other certain embodiments of this aspect, the method results in inhibition of bone resorption.
In yet another aspect, the invention provides methods for identifying an agent that modulates the expression or activity of osteoactivin nucleic acid molecules or proteins.
As used herein, an xe2x80x9cagentxe2x80x9d is a candidate or test compound (e.g., a protein, peptide, peptidomimetic, peptoid, small molecule or other chemical entity) which modulates the expression or activity of the osteoactivin nucleic acid molecule or protein. By xe2x80x9cmodulating the expression or activity of the osteoactivin nucleic acid molecule or proteinxe2x80x9d is meant a compound or molecule that has a stimulatory or inhibitory effect on, for example, osteoactivin expression or osteoactivin activity.
The method uses cells capable of expressing a gene under the control of the regulatory element(s) of an osteoactivin gene. Such cells include those which are capable of expressing an endogenous osteoactivin gene (e.g., an osteoblast cell line) or a cell transfected with a transgene comprising an osteoactivin regulatory element (e.g., an osteoactivin promoter) fused to a nucleic acid sequence encoding a polypeptide (e.g., an osteoactivin protein or a reporter protein), such that the osteoactivin gene regulatory element controls expression of the coding sequence.
In a preferred embodiment, the method uses host cells transfected with a nucleic acid comprising an osteoactivin regulatory element fused with nucleic acid sequence encoding a reporter protein. The preferred osteoactivin regulatory element comprises sequences spanning from just upstream of the ATG start site to 8-10 kb upstream of the ATG start site. The method comprises culturing separate samples of cells in the presence and absence of an agent in a suitable culture medium, wherein said cells express a gene under the control of an osteoactivin regulatory element; and measuring and comparing the levels of expression of said gene from said samples of cells cultured in the presence and absence of agent.
In still another aspect, the invention provides assays for determining the presence or absence of a genetic alteration in an osteoactivin polypeptide or in a nucleic acid encoding an osteoactivin protein. One embodiment of the invention is an assay for diagnosing osteopetrosis in a mammal suspected of suffering from osteopetrosis, comprising: (a) measuring the level of osteoactivin protein expression in a biological sample from said mammal; and (b) comparing said level of osteoactivin protein expression to a level of osteoactivin protein expression in a biological sample from a control.
The term xe2x80x9cbiological samplexe2x80x9d includes tissues, cells, and biological fluids isolated from a mammal, as well as tissues, cells and fluids present within a mammal.
In another aspect, a method for diagnosis of osteopetrosis in a mammal is provided. In this method, the level of osteoactivin in the mammal is measured and compared with the level of osteoactivin expressed in a control mammal which does not suffer from osteopetrosis, wherein increased expression in (a) compared to (b) is indicative of osteopetrosis in the mammal in (a).
It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory, and are intended to provide further explanation of the invention claimed.